Data from: Short-term spatial niche partitioning between the larger grain borer and the maize weevil in grain columns with implications for management of stored maize

The invasive Prostephanus truncatus, the larger grain borer, and Sitophilus zeamais, the maize weevil co-occur in many regions of the world. While competition between these two species has been studied extensively, there is little information on spatial partitioning in bulk storage of grain. To evaluate potential overlap in realized niche, we evaluated the short-term niche partitioning behavior of P. truncatus and S. zeamais in monolayers of maize alone or together for 1 d compared to 7 d. We evaluated competition under three different densities, namely 10-20, 75-150, and 150-300 insects/kg for P. truncatus and S. zeamais. The monolayers were equally divided into 24 zones to track location the abundance of insects and damage on maize. We found that both species generally aggregated together and were correlated to the same location as heterospecifics. After 1 d, most of the insects for both species were near the top of the monolayer, but by 7 d, most individuals were in the bottom of the monolayers. In monolayers, when alone, P. truncatus created a clear path of destruction to the bottom of the monolayer, but when S. zeamais was present, damage was lessened and shifted upwards in the grain column. In an olfactometer assay, P. truncatus preferred maize odors, while S. zeamais exhibited no preference among maize, conspecifics, and heterospecifics. In evaluating relative emissions, each of these treatments emitted unique odors but with significant overlap. These data may improve targeting of pest control tactics by identifying the position of these insects in the grain mass. Monolayers: Untreated, clean, and uninfested yellow maize was used in the experiments. Grain was sourced from a local farmer in northeast Kansas, US and frozen for 72 h prior to use to ensure no prior insect infestation was present. Monolayers were created with two sheets of glass cut to 75 x 27 cm (H:W). A spacer was then created using acrylic cut into 27-cm long x 1.5 cm width strips to form a barrier at the edges of the monolayer and to create a sufficient gap for a single kernel layer of grain between the two sheets of glass. The sheets of glass were taped with packing tape to ensure all gaps were adequately sealed to prevent insects from escaping and then held firmly in place along the edges of the glass sheets by binder clips (5.08 cm wide with 1.59 cm capacity). Before assembly, uninfested maize was uniformly and tightly laid out on one of the sheets of glass before the other one was added with spacers. Each assembled monolayer, consisting of two sheets of glass and spacers, was inserted into a wooden frame block (7.5 x 14 x 38 cm, H:W:L) with a gap just wide enough to accommodate the monolayer (~1.75 cm) and tightened with screws and wingnuts for foundational support. In addition, binder clips (similarly sized to above) were used to firmly attach it in place (Supplemental Figure 1A). Each sheet of glass was marked with 24 zones from top to bottom (8 rows and 3 columns of intersecting zones) (Supplemental Figure 1B). Zones were numbered from 1 to 24 starting in the top left corner of the monolayer. Once the insects were placed into the central top zone, the top of the monolayer was sealed with wire mesh to prevent insects from escaping at the top while also allowing air exchange. Treatments in Monolayer Assay: There were three insect densities and three species treatments. The three insect densities were 10, 75, or 150 insects of either species. The species treatments were either pure colonies of P. truncatus or S. zeamais or mixed species communities (both P. truncatus and S. zeamais together). This was an additive design, which has shown utility in the past in evaluating stored product insects (Mallqui et al., 2013). Insects were released simultaneously into the central top square of the monolayer and allowed to disperse for either one or seven days. Assays were run in an environmental chamber with constant temperature 27.5 +/- 0.1C and 65 +/- 0.3% RH at 14:10 L:D photoperiod. After the time period, each monolayer was taken apart carefully and the grain and insects from each zone were placed into separate bags and frozen to stop insect damage, movement, and growth. There were three replicates for each treatment combination. After all the samples were collected and frozen, each zone was carefully assessed for the abundance of each species and weight of grain damage by each species. Olfactometer Experiment: A total of 385 g of clean grain was placed in mason jars and the three treatments from the monolayer experiments were replicated. For the first, 200 P. truncatus were placed into a jar, for the second, 200 S. zeamais were placed into a jar, and for the third 100 of each species were placed into the same jar. The jars remained untouched for two weeks. After two weeks the insects were sifted out (1.41 x 1.41 mm sieve placed on top of a 2 x 2 mm mesh sieve) and frozen. The grain treatments were then used in a 4-way, still-air olfactometer (described in Ponce et al. 2022). Briefly, the apparatus consisted of a central release chamber of glass (12.1 cm x 9 cm H:D) with the bottom etched by acid connected by four passageways (3.5 cm x 2.5 cm L:D) spaced at 90 deg angles to the odor chambers (11.5 cm x 7 cm H:D). Passageways ended in a porous glass barrier that allowed diffusion of volatiles but prevented entry by insects in the first round of testing, and which had two equally spaced small holes. The odor chamber and release chamber were connected with a glass tube (7.5 cm x 2.2 L:D) attached on both sides with a PTFE (polytetrafluoroethylene) screw cap and septum. A total of 10 g of each of the three treatments were placed into the olfactometer odor chambers, while one was left empty as a blank control. Individual insects were then placed in the center of the release chamber of the olfactometer and given 5 min to make a decision. A decision was recorded when the insect moved 2.54 cm up the passageway leading to one of the treatments. Individuals that did not respond within 5 min were classified as non-responders and excluded from the final data analysis. The olfactometer was rotated by 90 deg after testing each insect to prevent any positional bias. After 10 replicate insects, the olfactometer was cleaned with odor-free detergent and water, followed by methanol and hexane, then completely allowed to dry before use. There were a total of n = 200 replicate individuals tested per insect species. Volatile Collection: Headspace collection was performed to determine relative differences in emissions among competitive treatments between S. zeamais and P. truncatus. Volatiles were collected for 3 h from each sample in autoclaved 250-mL media bottles (Corning Inc., Corning, NY, USA) with PTFE-lined septa, using 100 um polydimethylsiloxane (PDMS) solid phase microextraction (SPME) fibers (Supelco, Bellefonte, PA, USA), including from the following treatments: 20 g of maize conditioned with P. truncatus for two weeks, 20 g of maize conditioned with S. zeamais for two weeks, 20 g of maize conditioned with both species for two weeks, or an empty volatile collection container as a negative control. In each case, the SPME fiber was conditioned in the GC (Agilent 7890B gas chromatograph) inlet for 5 min at 250C prior to collecting samples. Media bottles were laid on their side, but grain never came into contact with SPME fiber. Directly after collection of samples, volatiles were desorbed on the GC-MS as below. An external standard curve of tetradecane was created by adding dilutions of tetradecane with dichloromethane in 1 uL aliquots added to 250-mL media bottles with a 2-uL syringe (Hamilton, Reno, NV, USA) corresponding to 10, 75, 190.5, and 381 ng of tetradecane. Headspace was collected in triplicate as above using identical SPME-GC-MS procedures. Glassware was wash with soap and water, and subsequently a combination of methanol, then hexane and placed in the drying oven (Heratherm, Thermofisher, Waltham, MA, USA). A total of n=8 replicate collections were done for each species and treatment combination. Gas chromatography coupled with mass spectrometry All headspace samples were manually injected into the inlet at 250C for 2.5 min and allowed to desorb from the SPME. Samples were run on an Agilent 7890B gas chromatograph (GC) equipped with an Agilent Durabond HP-5 column (30 m length, 0.250 mm diameter and 0.25 um film thickness) with He as the carrier gas at a constant 5 mL/min flow and 39 cm/s velocity, which was coupled with an Agilent 5997B mass spectrometer (MS) single quadrupole detector. The compounds were run under splitless mode. The oven program began at 30C for 1 min followed by 10C/min ramping to 300C over 26.5 min, and subsequently held for 4 min at 300C. After a solvent delay of 4 min, mass ranges between 50 and 550 atomic mass units were scanned. Preliminary identification of compounds from representative chromatograms were obtained by comparing sample spectral data with the NIST 17 library.